Figure 6.

Sema4A suppresses VEGF₁₆₅-mediated signaling. (A–D) HUVECs exposed for 5 h to control medium or Sema4A-Fc (50 nM) were further cultured in the absence or presence for VEGF₁₆₅ (50 ng/ml) for the indicated times, after which they were lysed for immunoprecipitation (IP) and/or immunoblot (Blot) assays. (A) Time course of tyrosine phosphorylation of VEGFR2 by VEGF₁₆₅. The tyrosine-phosphorylated VEGFR2 was immunoprecipitated using anti-VEGFR2 antibody and detected using anti-phospho-tyrosine antibody (4G10). To ensure comparable loading, total VEGFR2 protein was immunoblotted with anti-VEGFR2 antibody. (B) Time course of Rac activation by VEGF₁₆₅. Rac1-GTP was measured in pull-down assays using the GST-fused CRIB domain of PAK. Total Rac1 protein was immunoblotted with anti-Rac1 antibody. (C) Time course of the phosphorylation of Akt by VEGF₁₆₅. Phosphorylated Akt and total Akt were immunoblotted with anti-phospho-Akt antibody and anti-Akt antibody, respectively. (D) Time course of the phosphorylation of Erk by VEGF₁₆₅. Phosphorylated Erk and total Erk were immunoblotted with anti-phospho-Erk antibody and anti-Erk antibody, respectively. (E) Micrographs of HUVECs in response to VEGF₁₆₅ (50 ng/ml) and Sema4A-Fc (50 nM). HUVECs coinfected with or without adenoviruses containing mutant Rac and GFP were cultured in the indicated reagent for 5 h, and then stained with phalloidin–rhodamine. HUVECs expressing mutant Rac were identified by the coexpression of GFP. VEGF-mediated protrusion of lamellipodia (arrowhead) was suppressed by Sema4A-Fc (asterisk) and by treatment with RacN17 (star). The suppressive effect of Sema4A-Fc was cancelled by treatment with RacV12 (arrow). (F) Effects of mutant Rac on VEGF-mediated HUVEC migration. Twenty-four hours after coinfection with adenoviruses containing mutant Rac and GFP, cells were used for migration assay in the presence of VEGF₁₆₅ (50 ng/ml) with or without Sema4A-Fc (50 nM). HUVECs expressing mutant Rac were identified by the coexpression of GFP. VEGF-mediated migration of GFP-positive HUVECs was suppressed by Sema4A-Fc and by RacN17. The suppressive effect of Sema4A-Fc was cancelled by treatment with RacV12. ^*P<0.05 versus cells treated with Sema4A-Fc in the presence of VEGF₁₆₅.